Evaluation of major types of Shiga toxin 2E-producing Escherichia coli bacteria present in food, pigs, and the environment as potential pathogens for humans

Abstract

Shiga toxin 2e (Stx2e)-producing strains from food (n = 36), slaughtered pigs (n = 25), the environment (n = 21), diseased pigs (n = 19), and humans (n = 9) were investigated for production of Stx2e by enzyme-linked immunosorbent assay, for virulence markers by PCR, and for their serotypes to evaluate their role as potential human pathogens. Stx2e production was low in 64% of all 110 strains. Stx2e production was inducible by mitomycin C but differed considerably between strains. Analysis by nucleotide sequencing and transcription of stx(2e) genes in high- and low-Stx2e-producing strains showed that toxin production correlated with transcription rates of stx(2e) genes. DNA sequences specific for the int, Q, dam, and S genes of the stx(2e) bacteriophage P27 were found in 109 strains, indicating cryptic P27-like prophages, although 102 of these were not complete for all genes tested. Genes encoding intimin (eae), enterohemorrhagic Escherichia coli hemolysin (ehx), or other stx(1) or stx(2) variants were not found, whereas genes for heat-stable enterotoxins STI, STII, or EAST1 were present in 54.5% of the strains. Seven major serotypes that were associated with diseased pigs (O138:H14, O139:H1, and O141:H4) or with slaughter pigs, food, and the environment (O8:H4, O8:H9, O100:H30, and O101:H9) accounted for 60% of all Stx2e strains. The human Stx2e isolates did not belong to these major serotypes of Stx2e strains, and high production of Stx2e in human strains was not related to diarrheal disease. The results from this study and other studies do not point to Stx2e as a pathogenicity factor for diarrhea and hemolytic uremic syndrome in humans.

FIG. 1.

A DNA segment corresponding to positions 16866 to 19250 of the bacteriophage P27 genome (accession no. AJ298298) was investigated in strains CB10282 (AM937001) and CB10284 (AM939641) (A) as well as in strains CB10394 (AM939642) and CB10402 (AM939643) (B). (A) The low-Stx2e-producing strains CB10282 and CB10284 differed in only four nucleotides, at positions 1523, 2003, 2045, and 2325, from the AJ299298 sequence. Nucleotide changes leading to alterations in the amino acid sequence are indicated by asterisks. (B) The high-Stx2e-producing strains CB10304 and CB10402 differed in two nucleotides (positions 1523 and 1795) in the 1,749-bp stretch between stxA_2e and L28. These strains, as well as the high Stx2e producers CB8771 (AM940005), CB7671 (AM940007), CB8770 (AM940006), and CB8810 (AM940004), shared only 88.1% genetic homology with AJ298298 in the 344-bp tRNA region located upstream of stxA_2e (for details, see the Clustal analysis in Fig. 2). The “unknown” 241-bp region upstream left of the tRNA region (identical in strains CB10304, CB10402, CB8771, CB7671, CB8770, and CB8810) does not show similarity to any sequence deposited in GenBank.

FIG. 2.

(a and b) Clustal analysis of the tRNA region present in high-Stx2e-producing strains CB7671 (accession no. AM940007) (a) and CB10394 (AM939642) (b) and identical sequences present in CB10402 (AM939643), CB8771 (AM940005), CB8770 (AM940006), and CB8810 (AM940004). (c) Sequence present in low-Stx2e-producing strains CB10282 (AM937001) and CB10284 (AM939641) and in the high Stx2e producer 2771/97, which harbors the stx_2e-carrying phage P27 (AJ298298). The positions of tRNA genes and the start of the stxA_2e gene are indicated by bold arrows. Nucleotide changes and gaps are indicated by frames at the corresponding positions.